title: Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation.
abstract: A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl- secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast, the soluble type I cGK was unable to activate CFTR in intact cells, although both cGK I and cGK II could phosphorylate CFTR in vitro. To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, we expressed cGK II or cGK I mutants possessing different membrane binding properties by using adenoviral vectors in a CFTR-transfected intestinal cell line, and we examined the ability of cGMP to phosphorylate and activate the Cl- channel. Mutation of the cGK II N-terminal myristoylation site (Gly2 --> Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membrane-anchoring domain of cGK II was fused to the N terminus of cGK Ibeta, acquired the ability to associate with the membrane and activate the CFTR Cl- channel. The potency order of cGK constructs for activation of CFTR (cGK II > membrane-bound cGK I chimer >> nonmyristoylated cGK II > cGK Ibeta) correlated with the extent of 32P incorporation into CFTR observed in parallel measurements. These results strongly support the concept that membrane targeting of cGK is a major determinant of CFTR Cl- channel activation in intact cells.
authors.affiliation: Departments of Biochemistry, Cardiovascular Research Institute COEUR, 3000 DR Rotterdam, The Netherlands. Vaandrager@bc1.fgg.eur.nl
authors.forename: A B
authors.initials: AB
authors.lastname: Vaandrager
authors.forename: A
authors.initials: A
authors.lastname: Smolenski
authors.forename: B C
authors.initials: BC
authors.lastname: Tilly
authors.forename: A B
authors.initials: AB
authors.lastname: Houtsmuller
authors.forename: E M
authors.initials: EM
authors.lastname: Ehlert
authors.forename: A G
authors.initials: AG
authors.lastname: Bot
authors.forename: M
authors.initials: M
authors.lastname: Edixhoven
authors.forename: W E
authors.initials: WE
authors.lastname: Boomaars
authors.forename: S M
authors.initials: SM
authors.lastname: Lohmann
authors.forename: H R
authors.initials: HR
authors.lastname: de Jonge
mesh_terms: D000818:Animals; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000201:enzymology; D018118:Chloride Channels / Q000502:physiology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D006801:Humans; D015640:Ion Channel Gating; D009226:Myristates; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins; D014162:Transfection
chemical_list: C505032:CFTR protein, human; D018118:Chloride Channels; D009226:Myristates; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017869:Cyclic GMP-Dependent Protein Kinases